tissue clearing of mouse kidneys for confocal microscopy

Marcaje fluorescente de glomérulos en riñones de ratón aclarados para microscopía

The number and volume of glomeruli are very important for the diagnosis and treatment of various kidney diseases1,2,3,4,5. The golden standard of glomeruli number estimation is the physical dissector/fractionator combination. However, this method requires special reagents and equipment, making it slow and expensive6,7,8,9. Biopsy provides a wealth of information, but obviously, this method is only suitable for rough estimations10,11. Medical imaging technologies, including magnetic resonance imaging (MRI), computed tomography (CT), and X-ray, are also widely used in glomerular detection12,13,14,15, but such technologies require bulky instruments. New methods, such as matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometer16 or the thick and thin section method17, have also been used in glomerular detection, though they remain tedious and laborious.
With the help of transparency technologies, it is possible to observe deeper depths and obtain richer and more complete information from thick tissues or even whole organs18,19,20,21,22,23. Therefore, transparency technologies have been widely used in kidney research24. The observation and detection of glomeruli in the cleared kidneys are also involved. However, these published articles either only briefly referred to glomerular detection25 or used difficult-to-achieve labeling methods such as transgenic animals26, self-produced dyes13, or high-concentration antibody incubation27 to label the glomeruli. In addition, although studies had analyzed glomeruli in cleared kidneys, the analyses were always limited13 or relied on analysis algorithms established by the authors themselves26.
We have previously demonstrated a more convenient way to label the glomeruli in mice kidneys28. By using Imaris, we found that glomeruli count, frequency, and volume could be quickly obtained. Thus, here we present a more accessible, comprehensive, and simplified set of methods to label and analyze the glomeruli of mice kidneys.

Autor destacado: Ayudar a la investigación en biología renal mediante el etiquetado de glomérulos en tejidos aclarados 

Un método eficiente y rápido para etiquetar y analizar glomérulos de ratón

Labeling and counting glomeruli using conventional methods can be complicated, tedious, time consuming, and bulky instrument are required. Our method offers an instant and intuitively accurate labeling method for analysis of the glomeruli within the entire kidney. Our technique is a quick, easy, accurate, and cheap method to analyze glomeruli in the entire kidney.It does not require any special equipment, so it can be carried out in any laboratory. This is a groundbreaking, rapid, and simple method to label glomeruli, allowing for an intuitive observation of all the glomeruli within the entire kidney. It holds great potential to assist researchers to make significant breakthroughs in the study of the kidney by providing information such as glomeruli quantity, volume, distribution, and more.On one hand, we're exploring the application of this glomeruli labeling method in clinical kidney disease research. On the other hand, we'll keep working on enhancing methods to label biotargets like blood vessels or specific proteins in transparent tissues, adjusting them to meet our research needs.

Watch this Scientific Journal Video about Tissue Clearing of Mouse Kidneys for Confocal Microscopy at JoVE.com

Tissue Clearing of Mouse Kidneys for Confocal Microscopy  

Limpieza tisular de riñones de ratón para microscopía confocal

Para comenzar, coloque los riñones de ratón disecados y fijados en PBS. Corta el riñón en secciones de un milímetro de grosor usando la matriz cerebral. Transfiera las rodajas a un 4% de paraformaldehído y fíjelas a cuatro grados centígrados durante la noche.Para la microscopía de lámina de luz, fije todo el riñón sumergiéndolo en PFA al 4% e incubándolo a cuatro grados centígrados durante 48 horas. Para la limpieza de tejidos, recoja las muestras de riñón en un tubo de centrífuga de 50 mililitros y luego límpielas en L cúbico agitando a 60 RPM a 37 grados Celsius. Reemplace la L cúbica de forma intermitente hasta que se logre una transparencia óptica satisfactoria.Lave las muestras tres veces en PBS agitándolas suavemente a temperatura ambiente durante una hora cada una. A continuación, aplique R cúbico a las muestras y colóquelas para agitarlas durante seis horas. Observe las muestras aclaradas directamente o guárdelas en tubos opacos llenos de R cúbico a temperatura ambiente durante una semana.

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Research

JoVE Journal

Biology

Tissue Clearing of Mouse Kidneys for Confocal Microscopy

272 vistas.  Sichuan University. Para comenzar, coloque los riñones de ratón disecados y fijados en PBS. Corta el riñón en secciones de un milímetro de grosor usando la matriz cerebral. Transfiera las rodajas a un 4% de paraformaldehído y fíjelas a cuatro grados centígrados durante la noche.Para la microscopía de lámina de luz, fije todo el riñón sumergiéndolo en PFA al 4% e incubándolo a cuatro grados centígrados durante 48 horas. Para la limpieza de tejidos, recoja las muestras de riñón en un tubo d...

Video: Tissue Clearing of Mouse Kidneys for Confocal Microscopy  

An Efficient and Fast Method for Labeling and Analyzing Mouse Glomeruli

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological imaging provides intuitive information; thus, it is of great significance to label and observe the glomeruli. However, the glomeruli observation methods currently in use require complicated operations, and the results may lose label details or three-dimensional (3D) information. The clear, unobstructed brain imaging cocktails and computational analysis (CUBIC) tissue clearing technology has been widely used in renal research, allowing for more accurate detection and deeper detection depth. We found that mouse glomeruli can be rapidly and effectively labeled by tail vein injection of medium molecular weight FITC-Dextran followed by the CUBIC clearing method. The cleared mouse kidney could be scanned by a light-sheet microscope (or a confocal microscope when sliced) to obtain three-dimensional image stacks of all the glomeruli in the entire kidney. Processed with appropriate software, the glomeruli signals could be easily digitized and further analyzed to measure the number, volume, and frequency of the glomeruli.

This study presents an easy-to-use, complete, and simple set of methods to label and analyze glomeruli from CUBIC-cleared mouse kidneys. Data such as glomerulus number and volume can be obtained easily and reliably using fluorescein isothiocyanate (FITC)-Dextran, light sheet fluorescence microscopy (LSFM), or common confocal microscopy and software such as Imaris.

The glomeruli are fundamental units in the kidney; hence, studying the glomeruli is pivotal for understanding renal function and pathology. Biological ...